This invention relates to compositions and methods for the in vitro culture, transformation, and regeneration of plants.
Genetic improvement of various crop species by genetic engineering has sometimes been hindered because techniques for in vitro culture, transformation, and regeneration of amenable cultivars are less effective with recalcitrant commercial cultivars.
The ability to genetically engineer monocots, including cereal crops, to improve their performance and pest-resistance or to enhance alternative uses is of great importance. The practical utility of stable transformation technologies is largely dependent on the availability of efficient methods for generating large numbers of fertile green plants from tissue culture materials.
Virtually all current genetic engineering technologies require that genes be delivered to cells grown in vitro. Most published methods for generating fertile transformed plants from cereals (e.g. rice, wheat, maize, oat, sorghum, triticale, barley and rye) utilize as initial explants the immature scutellum of the embryo or microspores directly or tissue derived from immature embryos or microspores. From these initial explants, cellular proliferation occurs. After selection or screening for transformants, plants are regenerated.
Five critical problems adversely impact the utility of these transformation methods, particularly monocot species such as cereals and grasses. The first is heritable variability, termed xe2x80x9csomaclonal variationxe2x80x9d which results from spontaneous and heritable genetic changes in cultured plant tissues. Somaclonal variation can adversely affect the field performance (e.g., height, yield, and seed weight) of tissue culture-derived plants. Somaclonal variation can also limit the use of transgenic plants for breeding, since introgression of transgenes from such plants into acceptable genetic backgrounds can require multiple cycles of hybridization and progeny analysis, particularly if one or more heritable mutations were closely linked to the transgene or to genes controlling other critical traits.
The second problem is related to the increase in the incidence of albino plants caused by, for example, the physiological and biochemical changes imposed by selection, by the increased time frame required for selection during transformation and/or with changes in genotype. With some genotypes published methods of transformation result in the selection of transformed callus that is either nonregenerable or regenerates albino plants.
The third problem is the genotype dependence of the in vitro culture response of various explants, e.g. scutellum- and microspore-derived tissues and the resulting difficulties in applying transformation methods developed for amenable genotypes to commercially important more recalcitrant genotypes.
The fourth problem is related to the instability of the introduced genes themselves or of the expression of the transgenes. In transgenic cereals the introduced gene is sometimes lost in subsequent generations and there can also be a loss of the ability of the plant to express a transgene.
The fifth problem is that transgenic plants produced by published methods are often polyploid and therefore cannot be propagated as the more desirable diploid varieties, further limiting the usefulness of transgenic plants in breeding programs.
Aspects of the in vitro culturing and/or transformation process are likely to be responsible for or related to these and other problems encountered in efforts to genetically engineer plant species, including monocots such as cereals and grasses. Most transformation protocols require that the target tissue undergo embryogenesis, which may include de-differentiation of a single original transformed cell before the sustained cell divisions that give rise to an embryo consisting mostly or entirely of cells that contain the introduced DNA. De-differentiation during in vitro culturing introduces stresses on the genome, causing modifications of the genome that are associated with somaclonal variation, including DNA methylation, point mutations, deletions, insertions, and the generation of gross cytogenetic abnormalities. These genomic modifications lead to subsequent phenotypic abnormalities and performance losses and may contribute to the other problems listed above.
Transformation methods using excised shoot apices have been previously described (see, for example, U.S. Pat. No. 5,164,310 to Smith et al.; Zhong et al., 1996). However, these methods have not proven to be effective for some monocots, including commercially important varieties of barley, oat and wheat. For example, until now there is no method known for transformation of the spring barley cultivar (Hordeum vulgare L.) Harrington, which is a widely grown two-rowed malting barley.
There is a need, therefore, for improved methods for plant transformation and regeneration, particularly for use with monocot species such as cereals and grasses.
The invention provides methods and compositions for plant transformation and regeneration that are applicable to a wide variety of monocots, including commercially important cereal genotypes that have proven difficult or impossible to transform and regenerate by previously available methods. These improved methods result in significantly higher regeneration frequencies, reduced somaclonal variation, improved transgene expression stability, and reduced albinism.
The invention is based on the discovery that transgenic plants produced by transformation of organogenic tissues, particularly tissues derived from shoot meristematic cells, are generally healthier than plants produced by conventional transformation of embryogenic tissues. In particular, transgenic plants produced by transformation methods provided by the invention may be more stable, exhibit fewer problems associated with methylation, show fewer mutations caused by somaclonal variation, and exhibit reduced albinism. In addition, the transformation methods disclosed are applicable to commercial varieties of monocots such as barley, wheat and oat that are recalcitrant to conventional transformation methods.
The transformation method disclosed relies on introducing the nucleic acid sequence (generally referred to as the xe2x80x9ctransgenexe2x80x9d) into shoot meristematic tissue that is typically derived from a shoot apex or a leaf base. This tissue requires little or no de-differentiation in order to regenerate plants that express the transgene. Thus, in contrast to embryogenic callus tissue (a conventional target for transformation), these meristematic tissues do not undergo significant de-differentiation in the transformation process. Rather, these cells require only a simple redirection of growth in order to produce whole transgenic plants. The present invention also provides plant growth media containing growth substrates (including suitable levels of plant hormones and other components) with which the efficient production and regeneration of this meristematic tissue can be achieved. In particular, the invention provides media suitable for the production of meristematic tissue that is highly amenable to transformation from cultivars of monocots that are otherwise recalcitrant to transformation.
In general terms, the transformation method provided by the invention comprises obtaining shoot meristematic tissue from the plant to be transformed. Any source of shoot meristematic tissue may be employed, including shoot meristematic tissue taken from shoot apices of embryos and seedlings or axillary or adventitious shoots.
The meristematic tissue is incubated in the light on a meristem proliferation medium (MPM) to induce production of adventitious meristematic cells, which are then used as the target for nucleic acid transformation. Transformation may be achieved by any effective means, including for example conventional particle bombardment. MPM promotes fast growth of meristematic cells without promoting shoot or root formation. Particular compositions of MPM that are provided by this invention include components such as maltose and copper that are important to the success of the transformation methods; these compositions are designated as MPM-MC. MPM-MC typically comprises plant auxin and cytokinin hormones, usually in a low auxin/high cytokinin ratio. Thus, MPM-MC typically includes from 0 mg/l to about 3 mg/L of an auxin and from about 1 mg/L to about 10 mg/l of a cytokinin. MPM-MC also includes an elevated level of copper, generally from about 0.1 xcexcM to about 50 xcexcM, and typically within the range of about 1 to about 10 xcexcM. In addition, maltose is generally used as a carbon/sugar source in MPM-MC medium, typically at a concentration of from about 20 g/L to about 60 g/L, and usually at about 30 g/L. Other carbon sources, such as sucrose, may be used in place of, or in combination with, maltose. The invention provides particular combinations of these MPM-MC components that are especially suitable for use in transforming monocot species that were previously not amenable to transformation.
Following introduction of the nucleic acid, the meristematic tissues are typically transferred to fresh MPM-MC and incubated in the light. Thereafter, a selection agent may be introduced to the culture medium in order to select for transformed meristematic cells and meristematic structures. Transformed cells and structures are identified by their enhanced growth on this selection medium compared to untransformed material, and are subsequently removed and transferred to a regeneration medium for rooting.
The disclosed methods may be used not only for wheat, barley and oat, but also for other monocots, such as rice, maize, sorghum, millet, rye, triticale, forage grass and turfgrass.
These and other aspects of the invention will become more apparent from the following detailed description.
I. Shoot Meristematic Tissue as a Target Tissue for Plant Transformation
The transformation method of the present invention is based on the introduction of nucleic acids into meristematic tissues derived from any suitable source including, but not limited to, shoot meristems and leaf base tissue. These cells appear to require only a simple redirection of cells in the tissues for the formation of shoots and plants to occur in culture, unlike cells derived from an immature embryo or microspore which require an apparent de-differentiation process. The determination that a particular tissue observed in culture is meristematic shoot tissue (rather than embryogenic tissue) may be made based on a molecular analysis of the expression of certain genes which, as described in section II and Example 1 below, are shown to be expressed primarily in meristematic tissue and not in embryogenic tissue. This molecular approach permits the scientists subjective visual determination of meristematic tissues to be verified objectively.
Following introduction of the nucleic acids into the target meristematic cells, incubation without selection permits the meristematic tissue to proliferate, and allows the transformed tissue to become established. Subsequent application of a selection agent permits the transformed tissue to be selected. After the tissue has been selected (generally through multiple transfers to fresh selection medium to insure that the tissue comprising the meristematic domes is uniformly transformed), plants are induced to develop by removing or reducing the levels of hormones (particularly auxins) in the culturing medium (i.e., transfer to regeneration medium, RM). Plants can also be induced earlier in the selection process and resulting plants can be screened for presence of a transgene in the germline, thereby reducing the amount of time the tissue is cultured.
The use of meristematic tissue has several advantages for transformation. In particular, the adverse effects of a callus phase are eliminated, possibly because de-differentiation of the tissue does not occur (or occurs to a lesser extent) than during callus formation. Rather, adventitious meristems likely arise from a simple redirection of cells in the meristem or leaf base and not from de-differentiation.
Methylation problems associated with transformation of embryogenic tissue are reduced. An examination of barley plants arising from tissue that was derived from either immature embryos or meristems showed those derived from in vitro cultured meristems have fewer methylation changes at the genomic level and exhibit less somaclonal variation than plants derived from embryogenic callus. In addition, no albino plants were produced from meristem cultures, even after subculturing in vitro for more than one year. There is typically also less polyploidy in the meristem-derived regenerated plants and improved transgene expression stability.
In addition, the methods of the present invention are effective with a wider variety of genotypes than other transformation techniques, since de-differentiation of plant tissues, which is more genotype-dependent, is not required.
The methods of the present invention do not require the work- and resource-intensive maintenance of plants grown under controlled growth conditions as donor material that is required for many other transformation methods. This is because meristematic tissue can be derived from the developing shoot apices obtained by germinating dry seeds. By the methods of the present invention, meristematic cells derived from vegetative shoot meristems or young leaf bases can be proliferated in vitro and manipulated to give rise to plants through direct organogenesis (vegetative shoot meristem formation), eliminating the need to maintain the tissue in a de-differentiated state, and thereby reducing genomic instability. In addition proliferating meristematic tissue can be maintained for long periods without reduction in regenerative capacity.
The methods of the present invention are applicable to any plant species, including both dicot and monocot species, including, but not limited to barley, oat, wheat, rice, rye, triticale and turf and forage grasses. The disclosed methods are particularly useful for transformation of commercial varieties of wheat, barley and oat (e.g., barley genotypes Harrington, Morex, Galena, Steptoe, Moravian II, wheat genotypes Yecora Rojo, Bobwhite, Karl and Anza and oat genotypes Garry Gerry, Prone, Porter, Pacer and Ogle) that are recalcitrant to transformation using published embryogenic callus approaches.
II. Molecular Markers for Direct Organogenesis or Shoot Meristem Formation from In Vitro Cultured Tissue
Many plant somatic cells are totipotent and typically undergo in vitro regeneration via adventitious shoot meristem formation/development or somatic embryo formation. Adventitious shoot meristem formation/development, or organogenesis, is the process by which totipotent cells or tissues produce a unipolar structure, the shoot or root (including a shoot or root meristem, respectively), the vascular system of which is often connected to the parent tissue. Organogenesis likely results from a switch in the developmental program of pre-existing meristematic cells. In contrast, somatic embryogenesis occurs when a bipolar structure containing a root and shoot axis with a closed independent vascular system is produced from certain cells in the scutellum of the immature embryo or with cultured microspores. Somatic embryogenesis likely results from de-differentiation followed by a re-engagement of the whole developmental process in which plant cells participate during normal development of the zygotic embryo. A single isolated cell is able to develop normally into a whole plant via either formation of shoot meristem or somatic embryo, suggesting that the developmental program for formation of a shoot meristem or somatic embryo is contained within the cell.
In order to distinguish between embryogenic and organogenic tissue in culture, it has been necessary to rely on histology or on gross morphological features that are difficult to distinguish. The present invention identifies molecular markers that can be used to distinguish embryogenic from organogenic tissues far more easily and reliably, e.g., using immunological, molecular or microscopy methods. Thus, it is shown that expression of the knotted gene (e.g., maize kn1), for example, can be used to distinguish a shoot meristem from an embryo and to characterize shoot meristem proliferation in oat, barley, orchard grass and maize. Using anti-KN1 antibodies to assess KN1 expression during adventitious shoot meristem formation in in vitro-proliferating axillary shoot meristems of maize, barley and oat, it is shown that adventitious shoot meristems appear to arise directly from meristematic cells in the enlarged meristematic domes formed during in vitro culturing that express KN1 or KN1 homolog(s). These results provide molecular evidence that the in vitro morphogenic route in the described culture system is organogenesis.
Molecular markers for meristematic cells such as expression of KN1 and cdc2Zm permit one to identify meristematic tissues that can be used as target tissues for transformation. These molecular markers can also be used to assess whether modifications to a plant cell culture protocol such as the protocols provided herein promotes organogenesis and thus the ability to regenerate fertile green plants from cells cultured using the modified protocol.
III. Plant Culture Media and Methods
Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided below, definitions of common terms in molecular biology may also be found in Rieger et al., 1991; and Lewin, 1994.
a. In Vitro Culture of Meristematic Transformed Plant Cells
Meristematic tissue is comprised of undifferentiated plant cells that are capable of repeated division to yield other meristematic cells as well as differentiated cells that elongate and further specialize to form structural tissues and organs of the plant. Mersitematic tissue for use in the transformation methods described herein may be obtained from the shoot apices of seedlings or plants, as well as leaf bases.
The media used for in vitro culture of shoot meristematic tissue to produce adventitious meristems and to regenerate transformed meristematic tissue contribute significantly to the successful production of fertile transgenic plants. In addition, selection of fast-growing tissue improves the long-term regenerability of the cultures.
b. Meristem Proliferation Medium (MPM)
Meristematic tissue isolated from a plant (e.g., shoot apices) is cultured on MPM medium, which promotes a fast growth rate and proliferation of meristematic cells without promoting shoot and root formation. In addition, following DNA introduction into meristematic tissue, the transformed tissues are incubated on MPM for a time sufficient for individual transformed cells to proliferate, thereby ensuring that a sufficient number of progeny cells are produced from each transformation event to increase the likelihood that the initial transformation event leads to the regeneration of a plant containing transformed tissue.
MPM preferably has a low auxin/high cytokinin ratio. Auxin levels in MPM are typically about 0 mg/L (no auxin) to about 3.0 mg/L. For barley and wheat, for example, the preferred levels are about 0 mg/L to about 0.5 mg/L. For oat, no auxin is needed for optimal results. Cytokinin levels in MPM are typically about 1 mg/L to about 10 mg/L. For barley, wheat and oat, for example, about 2 mg/L to about 4 mg/L are preferred. Cytokinins may improve regenerability and reduce the incidence of albinism. The optimal level of cytokinin (and particularly the optimal ratio of auxin to cytokinin) depends on the genotype and the species being transformed.
Any well-known auxin or cytokinin may be used in MPM or regeneration medium (RM). Auxins include, but are not limited to, dichlorophenoxyacetic acid [2,4-D], dicamba, indoleacetic acid, picloran and naphthalenacetic acid. 2,4-D is preferred for barley, wheat and oat. Cytokinins include, but are not limited to, 6-benzylaminopurine [BAP], kinetin, zeatin, zeatin riboside, and N6-(2-isopentenyl)adenine (2iP). BAP and 2iP are typically employed for barley transformation, particularly BAP. A particular genotype or species may respond optimally to a specific phytohormone.
MPM-MC refers to the particular formulation of MPM used in certain aspects of the invention. MPM-MC is formulated with hormones as described above, and is supplemented with maltose and copper. MCM-MC contains copper generally at a concentration of at least 0.1 xcexcM (the level in typical plant growth media, such as MS medium), and more typically at least 10-100 fold higher, i.e. from about 1 to about 10 xcexcM. In certain formulations, MPM contains even higher levels of copper, for example up to about 50 xcexcM. Optimal copper levels vary with the genotype and species. The term xe2x80x9ccopperxe2x80x9d is intended to include any well-known nutritional source of copper for plant culture media, e.g., cupric sulfate.
In addition, MPM also includes a sugar/carbon source, generally at about 20 g/L to about 60 g/L, with about 30 g/L being typical. In MPM-MC maltose is the preferred carbon/sugar source, particularly for recalcitrant barley and wheat genotypes such as Harrington (barley) and Anza and Yecora Rojo (wheat) although sucrose or other conventional carbon sources for plant tissue culture can also be used (e.g., with barley genotype Golden Promise and oat).
Maltose and elevated copper levels were tested separately and in combination in various formulations of MPM to observe their effects on in vitro culture of adventitious meristems. In some barley and wheat genotypes, the combination of maltose and elevated copper levels was critical for the successful long-term proliferation of shoot meristematic tissue and dramatically improved the shoot meristem proliferation efficiency in Harrington (in which 80-90% of meristems cultured gave rise to proliferating adventitious meristematic tissue), in Crystal (50-70%), and in Morex (30-40%). Without the combination of maltose and elevated copper levels, none of the barley meristems gave rise to long-term cultures. Maltose alone reduced the production of brownish tissue, which had a negative effect on in vitro growth of the meristems, and elevated copper alone promoted plant development. However, the combination of maltose and copper was necessary to produce long-term regenerable meristematic tissues. In addition selection of fast-growing tissue was also important for obtaining long-term regenerative cultures.
As discussed in the Examples below, optionally MPM can be supplemented with a conventional osmoticum for a short time (e.g., about 4 hours) prior to (and, optionally, for a short period after) microprojectile bombardment. For example, the MPM can be supplemented with equimolar mannitol and sorbitol to give a final concentration of 0.4 M. However, good results have also been obtained when such an osmoticum was not included in MPM prior to (or after) bombardment.
As noted above, the methods and media described herein can be used to produce and maintain adventitious meristematic tissue for long periods of time. To maintain adventitious meristematic tissue, it is generally divided into smaller pieces (e.g., pieces of about 3 to 5 mm for barley) and subcultured, i.e., transferred to fresh medium, at regular intervals to promote optimal growth rates.
If a selectable marker is used to select for transformed tissues, the meristematic tissues may be initially cultured after transformation without selection in order to allow for the proliferation of transformed cells in the absence of dead or dying cells resulting from the selection agent. The optimal period for proliferation without selection varies with the species. After this period, selection can be applied to select for transformed cells. Selection can be accomplished by adding a selection agent to the culture medium for which the foreign DNA in transformed cells confers resistance (assuming that a selectable marker is included on the foreign DNA). Putative transformants are identified by their faster growth on the selective medium relative to nontransformed tissue. Screenable markers (e.g., green fluorescent protein and xcex2-glucuronidase) can also be used to identify transformed tissue.
Transformed tissues are generally maintained under light (for barley and wheat, approximately 10-30 xcexcEinsteins). The use of light reduces or eliminates the regeneration of albino plants and improves regenerability.
As used herein, xe2x80x9cplant culture mediumxe2x80x9d refers to any medium used in the art for supporting viability and growth of a plant cell or tissue, or for growth of whole plant specimens. Such media commonly include defined components including, but not limited to: macronutrient compounds providing nutritional sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, and iron; micronutrients, such as boron, molybdenum, manganese, cobalt, zinc, copper, chlorine, and iodine; carbohydrates (preferably maltose for barley, although sucrose may be better for some species); vitamins; phytohormones; selection agents (for transformed cells or tissues, e.g., antibiotics or herbicides); and gelling agents (e.g., agar, Bactoagar, agarose, Phytagel, Gelrite, etc.); and may include undefined components, including, but not limited to: coconut milk, casein hydrolysate, yeast extract, and activated charcoal. The medium may be either solid or liquid, although solid medium is preferred.
Any conventional plant culture medium can be used as a basis for the formulation of MPM and RM when appropriately supplemented as described herein. In addition to the plant culture media discussed in the Examples below (e.g., MS medium and FHG medium), a number of such basal plant culture media are commercially available from Sigma (St. Louis, Mo.) and other vendors in a dry (powdered) form for reconstitution with water.
c. Regeneration Medium.
xe2x80x9cRegeneration mediumxe2x80x9d (RM) promotes differentiation of totipotent plant tissues into shoots, roots, and other organized structures and eventually into plantlets that can be transferred to soil. Auxin levels in regeneration medium are reduced relative to MPM or, preferably, auxins are eliminated. It is also preferable that copper levels are reduced (e.g., to levels common in basal plant culture media such as MS medium). It is preferable to include a cytokinin in RM, as cytokinins have been found to promote regenerability of the transformed tissue. However, regeneration can occur without a cytokinin in the medium. Typically, cytokinin levels in RM are from about 0 mg/L to about 4 mg/L. For barley and wheat, about 2 mg/L of a cytokinin is preferred, and the preferred cytokinin is BAP. RM also preferably includes a carbon source, preferably about 20 g/L to about 30 g/L, e.g., either sucrose or maltose (there is no preference for maltose for RM).
Optionally, one may employ a conventional shooting medium to promote shoot regeneration from meristematic structures and/or a conventional rooting medium to promote root formation. For example, MS basal medium supplemented with IBA (e.g., 0.5 mg/L) can be used to induce root formation, if necessary. Root induction is preferred for corn but appears to generally be unnecessary with oat and barley. Depending upon the genotype, different levels of an auxin and cytokinin (i.e., a different auxin/cytokinin ratio) provide optimal results. Conventional shooting and rooting media are considered regeneration media.
Any well-known regeneration medium may be used for the practice of the methods of the present invention. For barley, FHG medium (Hunter, 1988, and described in Kasha et al., 1990) can be used, for example.
d. Introduction of Nucleic Acids
A number of methods can be used to introduce nucleic acids into the meristematic cells, including particle bomardment. Particle bombardment has been employed for transformation of a number of plant species, including barley (see, e.g., Wan and Lemaux, 1994, and BioRad Technical Bulletin 2007) and corn (see, e.g., Gordon-Kamm et al., 1990, Wan et al., 1995), for example. Successful transformation by particle bombardment requires that the target cells are actively dividing, accessible to microprojectiles, culturable in vitro, and totipotent, i.e., capable of regeneration to produce mature fertile plants. As described herein, a meristematic tissue (including, but not limited to a vegetative shoot meristem, such as an apical meristem from primary or axillary shoots, or a young leaf base) is cultured in vitro to caused to formation of adventitious meristems, and the adventitious meristem cells are the target for bombardment.
Microprojectile bombardment can be accomplished at normal rupture pressures, e.g., at about 1100 psi, although lower rupture pressures can be used to reduce damage of the target tissue, e.g., about 600 to 900 psi. It has been found that meristematic tissues recover better from the tissue damage caused by bombardment than callus tissue, permitting higher rupture pressures to be used.
In addition to particle bombardment, conventional methods for plant cell transformation may be used, including but not limited to: (1) Agrobacterium-mediated transformation, (2) microinjection, (3) polyethylene glycol (PEG) procedures, (4) liposome-mediated DNA uptake, (5) electroporation, and (6) vortexing with silica fibers.
e. Definitions and Explanations of Terms Used
The following definitions and explanations are provided to facilitate understanding of the invention.
Plant: The term xe2x80x9cplantxe2x80x9d encompasses transformed plants, progeny of such transformed plants, and parts of plants, including reproductive units of a plant, fruit, flowers, seeds, etc. The transformation methods and compositions of the present invention, are particularly useful for transformation of cereal genotypes that are recalcitrant to other transformation methods. Such cereals include barley (e.g., genotypes Morex, Harrington, Crystal, Stander, Moravian III, Galena, Salome, Steptoe, Klages and Baronesse), wheat (e.g., genotypes Yecora Rojo, Bobwhite, Karl and Anza) and oat (e.g., genotypes Garry Gerry, Prone, Porter, Pacer and Ogle). Other species of monocotyledonous and dicotyledonous plants may also be transformed using the disclosed methods.
Reproductive unit: A reproductive unit of a plant is any totipotent part or tissue of the plant from which one can obtain progeny of the plant, including, for example, seeds, cuttings, tubers, buds, bulbs, somatic embryos, microspores, cultured cells (e.g., callus or suspension cultures), etc.
Isolated: An isolated nucleic acid is one that has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA. The term also embraces recombinant nucleic acids and chemically synthesized nucleic acids.
Operably Linked: Nucleic acids can be expressed in plants or plant cells under the control of an operably linked promoter that is capable of driving expression in a cell of a particular plant. A first nucleic-acid sequence is operably linked with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, to join two protein coding regions to produce a hybrid protein.
Recombinant: A recombinant nucleic acid is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by conventional genetic engineering techniques.
Transformed; Transgenic: A cell, tissue, organ, or organism into which a foreign nucleic acid, such as a recombinant vector, has been introduced is considered xe2x80x9ctransformedxe2x80x9d or xe2x80x9ctransgenic,xe2x80x9d as is progeny thereof in which the foreign nucleic acid is present. A transformed tissue or plant may include some cells that are not transformed, i.e., may be chimaeric, comprising transformed and untransformed cells. Such chimaeric tissues may be used to regenerate transformed plants, and may be advantageous for this purpose since less in vitro propagation and selection will be required to produce chimaeric tissues than tissues in which 100% of the cells are transformed. Regeneration of chimaeric tissues will generally give rise to chimaeric plants, i.e., plants comprised of transformed and non-transformed cells. Reproduction of these chimaeric plants by asexual or sexual means may be employed to obtain plants entirely comprised of transformed cells.
xe2x80x9cForeignxe2x80x9d nucleic acids are nucleic acids that would not normally be present in the host cell, particularly nucleic acids that have been modified by recombinant DNA techniques. The term xe2x80x9cforeignxe2x80x9d nucleic acids also includes host genes that are placed under the control of a new promoter or terminator sequence, for example, by conventional techniques.
Vectors, Transformation, Host cells: Nucleic acids can be incorporated into recombinant nucleic-acid constructs, typically DNA constructs, capable of being introduced into and replicating in a host cell. Such a construct preferably is a vector that includes sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell (and may include a replication system, although direct DNA introduction methods conventionally used for monocot transformation do not require this).
For the practice of the present invention, conventional compositions and methods for preparing and using vectors and host cells are employed, as discussed, inter alia, in Sambrook et al., 1989, or Ausubel et al., 1992.
A number of vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., 1987, Weissbach and Weissbach, 1989, and Gelvin et al., 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5xe2x80x2 and 3xe2x80x2 regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Examples of constitutive plant promoters useful for expressing genes in plant cells include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, maize ubiquitin (Ubi-1) promoter, rice actin (Act) promoter, nopaline synthase promoter, and the octopine synthase promoter. A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals also can be used for expression of foreign genes in plant cells, including promoters regulated by heat (e.g., heat shock promoters), light (e.g., pea rbcS-3A or maize rbcS promoters or chlorophyll a/b-binding protein promoter); phytohormones, such as abscisic acid; wounding (e.g., wunI); anaerobiosis (e.g., Adh); and chemicals such as methyl jasminate, salicylic acid, or safeners. It may also be advantageous to employ well-known organ-specific promoters such as endosperm-, embryo-, root-, phloem-, or trichome-specific promoters, for example.
Plant expression vectors optionally include RNA processing signals, e.g., introns, which may be positioned upstream or downstream of a polypeptide-encoding sequence in the transgene. In addition, the expression vectors may also include additional regulatory sequences from the 3xe2x80x2-untranslated region of plant genes, e.g., a 3xe2x80x2 terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3xe2x80x2 terminator regions.
Such vectors also generally include one or more dominant selectable marker genes, including genes encoding antibiotic resistance (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, paromomycin, or spectinomycin) and herbicide-resistance genes (e.g., resistance to phosphinothricin acetyltransferase or glyphosate) to facilitate manipulation in bacterial systems and to select for transformed plant cells.
Screenable markers are also used for plant cell transformation, including color markers such as genes encoding xcex2-glucuronidase (gus) or anthocyanin production, or fluorescent markers such as genes encoding luciferase or green fluorescence protein (GFP).
The invention is illustrated by the following Examples.